A single activity carboxyl methylates both farnesyl and geranylgeranyl cysteine residues.

Members of the Ras superfamily of small GTP-binding proteins, gamma-subunits of heterotrimeric G proteins and nuclear lamin B are subject to a series of post-translational modifications that produce prenylcysteine methylester groups at their carboxyl termini. The thioether-linked polyisoprenoid substituent can be either farnesyl (C15) or geranylgeranyl (C20). Small molecule prenylcysteine derivatives with either the C15 or C20 modification, such as N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), S-trans,trans-farnesylthiopropionate (FTP), as well as the corresponding geranylgeranyl derivatives (AGGC and GGTP) are substrates for the carboxyl methyltransferase. Saccharomyces cerevisiae ste14 mutants that lack RAS and a-factor carboxyl methyltransferase activity are also unable to methylate farnesyl and geranylgeranylcysteine derivatives. Moreover, C20-substituted cysteine analogs directly compete for carboxyl methylation with the C15-substituted cysteine analogs and vice versa. Finally, AGGC is even more effective than AFC as an inhibitor of Ras carboxyl methylation, despite the fact that Ras is methylated at a farnesylcysteine rather than a geranylgeranylcysteine residue.     

A novel protein complex that regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5‐methylcytosine DNA glycosylase/lyase ROS1 initiates a base‐excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1 000s of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl‐DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo. Loss‐of‐function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.     

Arabidopsis Trithorax histone methyltransferases are redundant in regulating development and DNA methylation

Although the Trithorax histone methyltransferases ATX1–5 are known to regulate development and stress responses by catalyzing histone H3K4 methylation in Arabidopsis thaliana, it is unknown whether and how these histone methyltransferases affect DNA methylation. Here, we found that the redundant ATX1–5 proteins are not only required for plant development and viability but also for the regulation of DNA methylation. The expression and H3K4me3 levels of both RNA-directed DNA methylation (RdDM) genes (NRPE1, DCL3, IDN2, and IDP2) and active DNA demethylation genes (ROS1, DML2, and DML3) were downregulated in the atx1/2/4/5 mutant. Consistent with the facts that the active DNA demethylation pathway mediates DNA demethylationmainly at CG and CHG sites, and that the RdDM pathway mediates DNA methylation mainly at CHH sites, whole-genome DNA methylation analyses showed that hyper-CG and CHG DMRs in atx1/2/4/5 significantly overlapped with those in the DNA demethylation pathway mutant ros1 dml2 dml3 (rdd), and that hypo-CHH DMRs in atx1/2/4/5 significantly overlapped with those in the RdDM mutant nrpe1, suggesting that the ATX paralogues function redundantly to regulate DNA methylation by promoting H3K4me3 levels and expression levels of both RdDM genes and active DNA demethylation genes. Given that the ATX proteins function as catalytic subunits of COMPASS histone methyltransferase complexes, we also demonstrated that the COMPASS complex components function as a whole to regulate DNA methylation. This study reveals a previously uncharacterized mechanism underlying the regulation of DNA methylation.     

Carboxyl methylation of 21-23 kDa membrane proteins in intact neuroblastoma cells is increased with differentiation

Evidence is presented for specific enzymatic methylation of 21-23 kDa membrane proteins in intact neuroblastoma N1E 115 cells, which is increased in dimethylsulfoxide-induced differentiated cells. Methylation of these proteins has characteristics typical of enzymatic reactions in which base labile volatile methyl groups are incorporated into proteins, consistent with the formation of protein carboxyl methylesters. However, these methylesters of the 21-23 kDa proteins are relatively stable compared to other protein carboxyl methylesters. The 3-fold increase in methylated 21-23 kDa proteins in the differentiated cells suggest biological significance in differentiation of the cell membranes.     

Targeting protein lysine methylation and demethylation in cancers

During the last decade, we saw an explosion of studies investigating the role of lysine methylation/demethylation of histones and non-histone proteins, such as p53, NF-kappaB, and E2F1. These 'Ying-Yang' post-translational modifications are important to fine-tuning the activity of these proteins. Lysine methylation and demethylation are catalyzed by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). PKMTs, PKDMs, and their substrates have been shown to play important roles in cancers. Although the underlying mechanisms of tumorigenesis are still largely unknown, growing evidence is starting to link aberrant regulation of methylation to tumorigenesis. This review focuses on summarizing the recent progress in understanding of the function of protein lysine methylation, and in the discovery of small molecule inhibitors for PKMTs and PKDMs. We also discuss the potential and the caveats of targeting protein lysine methylation for the treatment of cancer.     

The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.     

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis

DNA methylation,a conserved epigenetic mark,is critical for tuning temporal and spatial gene expression.The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1(ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci.However,how ROS1 is recruited to specific loci is not well understood.Here,we report the discovery of Arabidopsis AGENET Domain Containing Protein 3(AGDP3) as a cellular factor that is required to prevent g...     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

The RNA N6-methyladenosine demethylase ALKBH9B modulates ABA responses in Arabidopsis

The mRNA modification N⁶-methyladenosine(m⁶A)plays vital roles in plant development and biotic and abiotic stress responses.The RNA m⁶A demethylase ALKBH9 B can remove m⁶A in alfalfa mosaic virus RNA and plays roles in alfalfa mosaic virus infection in Arabidopsis.However,it is unknown whether ALKBH9 B also exhibits demethylation activity and has a biological role in endogenous plant mRNA.We demonstrated here that mRNA m⁶A modification is in...     

The carboxyl methyltransferase modifying G proteins is a metalloenzyme.

The prenylated protein carboxyl methyltransferase (PPMT) catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as the small monomeric G proteins and the gamma subunits of heterotrimeric G proteins. Here we report that both membrane-bound PPMT from rat kidney and the recombinant bacterially expressed form of the enzyme required divalent cations for catalytic activity. Unlike EDTA and EGTA, the metal chelator 1,10-phenanthroline strongly inhibited the PPMT activity of kidney intracellular membranes in a dose- and time-dependent manner. 1,10-Phenanthroline was found to inhibit the methylation of the prenylcysteine analog N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, a synthetic substrate for PPMT, with an IC(50) of 2.2 mM. Gel electrophoretic analysis demonstrated that 1,10-phenanthroline almost totally abolished the labeling of methylated proteins in kidney intracellular membranes. Immunoblotting analysis showed that one of the two major peaks of (3)H-methylated proteins in intracellular membranes comigrated with the small G proteins Ras, Cdc42, RhoA, and Rab1. In addition, the methylation of immunoprecipitated Ras and RhoA from kidney intracellular membranes was strongly inhibited when 1,10-phenanthroline was present. Treatment of kidney intracellular membranes with 1,10-phenanthroline increased the proteolytic degradation of PPMT by exogenous trypsin, compared to untreated membranes. We conclude from these data that metal ions are essential for the activity and the stabilization of PPMT. The finding that PPMT is a metalloenzyme may provide new insights into the functions played by this methyltransferase in signal transduction processes.     

P53蛋白赖氨酸甲基化与去甲基化

P53作为肿瘤抑制因子和转录调节因子在控制细胞周期、凋亡和DNA修复方面发挥重要作用。P53蛋白的稳定性和转录激活活性的调节主要依赖磷酸化、乙酰化、泛素化等多种翻译后修饰。最近研究发现一些组蛋白赖氨酸甲基转移酶和去甲基化酶可使P53蛋白C-端赖氨酸残基发生甲基化或去甲基化,调节P53蛋白的稳定性和转录激活活性。甲基化和去甲基化与其它翻译后修饰相互作用构成“P53密码”调节P53蛋白功能。     

RNA-directed DNA methylation has an important developmental function in Arabidopsis that is masked by the chromatin remodeler PICKLE

In Arabidopsis, RNA‐directed DNA methylation (RdDM) is required for the maintenance of CHH methylation, and for de novo methylation in all (CG, CHG, and CHH) contexts, but no obvious effect of RdDM deficiency on plant development has been found to date. We show that the combination of mutations in the chromatin remodeler PKL and RdDM components results in developmental alterations, which appear in a SUPPRESSOR OF DRM1 DRM2 CMT3 (SDC)‐dependent manner.     

N4‐methylcytidine ribosomal RNA methylation in chloroplasts is crucial for chloroplast function,development, and abscisic acid response in Arabidopsis

Although the essential role of messenger RNA methylation in the nucleus is increasingly understood, the nature of ribosomal RNA (rRNA) methyltransferases and the role of rRNA methylation in chloroplasts remain largely unknown. A recent study revealed that CMAL (for Chloroplast mr aW‐ Like) is a chloroplast‐localized rRNA methyltransferase that is responsible for N4‐methylcytidine (m⁴C) in 16S chloroplast rRNA in Arabidopsis thaliana. In this study, we further examined the role of CMAL in chloroplast biogenesis and function, development, and hormone response. The cmal mutant showed reduced chlorophyll biosynthesis, photosynthetic activity, and growth‐defect phenotypes, including severely stunted stems, fewer siliques, and lower seed yield. The cmal mutant was hypersensitive to chloroplast translation inhibitors, such as lincomycin and erythromycin, indicating that the m⁴C‐methylation defect in the 16S rRNA leads to a reduced translational activity in chloroplasts. Importantly, the stunted stem of the cmal mutant was partially rescued by exogenous gibberellic acid or auxin. The cmal mutant grew poorer than wild type, whereas the CMAL‐overexpressing transgenic Arabidopsis plants grew better than wild type in the presence of abscisic acid. Altogether, these results indicate that CMAL is an indispensable rRNA methyltransferase in chloroplasts and is crucial for chloroplast biogenesis and function, photosynthesis, and hormone response during plant growth and development.     

The methyl-accepting chemotaxis proteins of E. coli: a repellent-stimulated, covalent modification, distinct from methylation

The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are integral membrane proteins that have been shown to undergo reversible methylation in response to the addition of attractants. We have shown that a second, rapid modification of MCPI and MCPII occurs, which is repellent-stimulated. This modification, which is not methylation, was detected because it causes a decrease in mobility of the MCPs on 7.5% SDS-polyacrylamide gels with a high acrylamide to bisacrylamide ratio. We have designated this modification as the CheB-modification, as it is dependent on the CheB gene product. The CheB-modification causes a decrease in the isoelectric point of MCPII by one or two charge groups. The CheB-modification is not necessary for the methylation, nor does it preclude methylation of the MCPs. Both the CheB-modified form and the unmodified, unmethylated forms of the MCPs are stable to treatment with base, which results in the hydrolysis of the methylesters (demethylation) of the MCPs. The potential role of CheB-modification in chemotaxis is discussed.